International Journal of Molecular Sciences Article Physiological Importance of Pectin Modifying Genes During Rice Pollen Development 1, 2, 1, 1 1 Yu-Jin Kim y , Ho Young Jeong y, Seung-Yeon Kang y, Jeniffer Silva , Eui-Jung Kim , Soon Ki Park 3, Ki-Hong Jung 1,* and Chanhui Lee 2,* 1 Graduate School of Biotechnology & Crop Biotech Institute, Kyung Hee University, Yongin 17104, Korea; [email protected] (Y.-J.K.); [email protected] (S.-Y.K.); jeniff[email protected] (J.S.); [email protected] (E.-J.K.) 2 Department of Plant & Environmental New Resources, College of Life Sciences, Kyung Hee University, Yongin 17104, Korea; [email protected] 3 School of Applied Biosciences, Kyungpook National University, Daegu 41566, Korea; [email protected] * Correspondence: [email protected] (K.-H.J.); [email protected] (C.L.); Tel.:+82-31-201-3474 (K.-H.J.) These authors contributed equally to this work. y Received: 23 May 2020; Accepted: 7 July 2020; Published: 8 July 2020 Abstract: Although cell wall dynamics, particularly modification of homogalacturonan (HGA, a major component of pectin) during pollen tube growth, have been extensively studied in dicot plants, little is known about how modification of the pollen tube cell wall regulates growth in monocot plants. In this study, we assessed the role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Both treatments led to a severe decrease in the pollen germination rate and elongation. Furthermore, using monoclonal antibodies toward methyl-esterified and de-esterified HGA epitopes, it was found that exogenous treatment of PME and Polyphenon 60 resulted in the disruption of the distribution patterns of low- and high-methylesterified pectins upon pollen germination and during pollen tube elongation. Eleven PMEs and 13 PME inhibitors (PMEIs) were identified by publicly available transcriptome datasets and their specific expression was validated by qRT-PCR. Enzyme activity assays and subcellular localization using a heterologous expression system in tobacco leaves demonstrated that some of the pollen-specific PMEs and PMEIs possessed distinct enzymatic activities and targeted either the cell wall or other compartments. Taken together, our findings are the first line of evidence showing the essentiality of HGA methyl-esterification status during the germination and elongation of pollen tubes in rice, which is primarily governed by the fine-tuning of PME and PMEI activities. Keywords: rice; pollen; pectin; pectin methylesterase; pectin methylesterase inhibitor; pollen tube growth 1. Introduction All plant cells are encased by walls with a distinct polysaccharide composition. Some specialized cells, including those of root hairs and pollen tubes, undergo rapid directional elongation [1–3]. Once differentiated, these types of cells depend upon the coordinated action of cell wall-modifying enzymes (CWMEs), which promote wall loosening and extensibility by disrupting chemical bonds or selectively removing specific polysaccharide side chains [4]. All higher plants contain a wide range of CWMEs, such as expansin, glycosyl hydrolases, and carbohydrate esterases and lyases [5,6]. Pectin is a polysaccharide found mainly in the middle lamella and primary cell wall, and plays a critical role in wall plasticity and cellular adhesion [7]. Compositional analysis of grass cell walls has revealed that pectin accounts for approximately 5% of the cell wall components [8,9]. Int. J. Mol. Sci. 2020, 21, 4840; doi:10.3390/ijms21144840 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 4840 2 of 16 Pectin has a galacturonic acid (GalUA)-rich backbone of four domains that can be distinguished based on their structure and substitution patterns: homogalacturonan (HGA), rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII), and xylogalacturonan (XGA) [6,9]. HGA, a linear polymer of (1,4)-linked-α-GalUA, is the predominant form of pectin and becomes heavily methyl-esterified at the C-6 carboxyl by pectin methyltransferases when in the Golgi apparatus [10]. Following this, it is transported and deposited into cell walls to connect to their matrices, wherein it undergoes further modifications depending on the cell type and its position in the pectin network. One of the best known types of modification is demethyl-esterification (selective removal of methyl groups) by pectin methylesterases (PMEs), which is believed to profoundly impact the wall’s mechanical properties in a variety ways. Thus, fine-tuning PME activities are imperative for fulfilling cellular functions. PME proteins are grouped into two types, depending on the presence of an N-terminal pro region, and labeled as type 1 (with pro region) and type 2 (without pro region) [11,12]. This N-terminal pro region appears to function as a self-inhibitory domain in the Golgi apparatus, which is removed proteolytically before being transported out by secretory vesicles [13]. In addition, the enzymatic action of PME is regulated by PME inhibitors (PMEIs), which belong to a large multigene-encoded protein family. Co-crystallization experiments using the tomato (Solanum lycoperscicum) PME and kiwi (Actinidia chinensis) PMEI complex have demonstrated that PMEIs bind specifically to the pectin-binding cleft of PME [14], and as such, it is generally believed that specific PME-PMEI pairs exist. A recent simulation analysis using Arabidopsis PME (AtPME3) and PMEIs (AtPMEI4 and AtPMEI9), which are coexpressed in roots, uncovered key residues playing a critical role in the specificity and pH-dependence of inhibitor binding [15]. Given that all higher plants possess PMEs and PMEIs, and that pectin HGA is found in most plant cell types, it is not surprising that misregulation of those genes can cause a wide range of developmental abnormalities. Changes in the transcription levels of specific PMEs and PMEIs, either through overexpression or knock-out approaches, strongly influence the degree of pectin methyl-esterification in certain cell types and developmental processes, including dark-grown hypocotyl elongation, pollen tube elongation, dormancy, germination, seed mucilage production and pathogenesis [16,17]. Importantly, several PME and PMEI isoforms show specific expression in pollen and pollen tubes in dicot plants, and Arabidopsis mutants with reduced PME activities have displayed severe defects in pollen tube growth and morphology [18]. Furthermore, adding PME or pectinase to a pollen germination medium resulted in abnormal growth of the pollen tube in Solanum chacoense [19]. Immunohistochemical studies in dicot plants have revealed that highly methyl-esterified HGA is prevalent in the apex of the pollen tube, whereas minimally methyl-esterified HGA is dominant in lateral region of the pollen tube [10]. The establishment of this disproportion is governed by the fine-tuning of PMEIs. Previously, we reported that the rice genome has 43 PMEs and 49 PMEIs [7,20]. Although it is well known that dynamic changes in the methyl-esterification of HGA by PME and PMEI enzymes are critical for proper pollen tube growth in dicot plants, the involvement of PME and PMEI in regulating monocot pollen tube development is still poorly understood. To investigate their importance in the development of rice pollen, we first assessed the effects of pollen tube growth following the addition of a PME enzyme and PME-inhibiting catechin extract (Polyphenon 60) to an in vitro pollen germination medium. Eleven pollen-specific PMEs and 13 PMEIs were identified in rice and enzymatic activities were measured. Our findings showed that spatial, post-translational control of PME activities by PMEI plays a key role in disproportional deposition of methyl-esterified HGA in the pollen tube walls of rice. 2. Results 2.1. The Effects of a PME and PME-Inhibiting Catechin Extract on In Vitro Pollen Germination in Rice Previous genetic and biochemical studies on dicot pollen development have shown that the degree of HGA methyl-esterification is one of the most critical determinants of pollen germination Int. J. Mol. Sci. 2020, 21, 4840 3 of 16 and tip elongation [3,11,21–23]. To directly determine if fine-tuning of HGA methyl-esterification status via PME and PMEI enzymatic activities is essential during rice pollen tube growth, we added various concentrations of commercial PME to an in vitro pollen germination medium, with the intention of disturbing the cellular balance and creating minimally methyl-esterified HGA across the pollen cell walls. In our experiment, about 80% of the rice pollens germinated within 20 min (Figure1A,D), and a decreased rate of pollen germination was observed in the PME supplemented medium (Figure1B,C). In addition, pollen tube growth was severely a ffected: while normal rice pollen tubes grew to an average length of 100 µm, the germinated pollen tubes ceased to grow beyond 50 µm in length at low concentrations of PME (Figure1D–F), and burst immediately at the highest concentration of PME (Figure1G, marked by red arrow). Nearly 50% of the pollen grains germinated at a concentration of 1 units mL 1 PME, nearly 2% germinated at a concentration of 3 units mL 1 · − · − PME, and no pollen germination was observed at higher concentrations of 4–15 unit mL 1 PME · − (Figure1N). To further verify that the degree of HGA methyl-esterification was a critical factor for pollen tube growth, a PME-inhibiting catechin extract (Polyphenon 60) was added to the germination medium [24,25]. Although epigallocatechin-3-gallate, the main component of Polyphenon 60, has been recently reported to affect broad ranges of plant physiology including regulation of jasmonic acid signaling and disease resistance, its specific inhibitory effect to PME has been well documented [26]. The amount of germination was slightly affected by the concentration of 0.5 mg mL 1 of the catechin · − extract (Figure1H), and decreased in a dose-dependent manner (Figure1H–J,O). Germination of the pollen tube decreased significantly following treatment with high concentrations of Polyphenon 60, compared to that of the control (Figure1I,J).